A cell membrane serves as an obstacle to permeability of proteins, nucleic acids, peptides and insoluble compounds into cells due to impermeability. This problem in intracellular drug transduction must be solved in fields of treatment, prevention and diagnosis of diseases. Generally, methods of permeating biologically active macromolecules into a cell membrane include electroporation, membrane fusion using a liposome, transfection using calcium phosphate, a cationic polymer such as polyethyleneimine (PEI) or DEAE-dextran, viral transfection, and single cell microinsertion. In addition, recently, for effective intracellular drug transduction, techniques of applying endocytosis of a drug delivery system such as nanoparticles have been used, but more studies on achieving effective intracellular drug delivery are needed due to a decrease in delivery efficiency caused by fast loss in an immune system, or steric hindrance caused by interaction with cells. Accordingly, recently, development of a method of delivering macromolecules such as proteins, nucleic acids, etc. through a biomembrane and a nuclear envelope of a cell without damage to a cell membrane has been continuously needed.
In 1988, the group of Green and Loewenstein (Green and Loewenstein. Cell 55, 1179-1188(188)) and the group of Frankel and Pabo (Frankel and Pabo. Cell 55, 1189-1193(188)) each discovered a transduction domain permeating a transcription-associated protein, Tat, of HIV-1, which is the virus that causes acquired immune deficiency syndrome (AIDS), through a cell membrane and trans-activating a viral gene. A Tat domain exhibiting cell permeability is a sequence of 48th to 57th basic amino acids (GRKKRRQRRR; SEQ ID NO: 57) of the Tat protein, and it was found that the sequence serves an important role in permeating the cell membrane (Vives et al., J. Biol. Chem. 272, 16010-16017 (1997); Futaki et al, J. Biol. Chem. 276, 5836-5840 (2001); Wadia, J. S. and S. F. Dowdy, Cum Opin. Biotechnolol. 13(1): 52-6 (2002); Wadia et al, Nature Medicine 10(3), 310-315(2004)).
As another example, Antennapedia homeodomain-derived penetratin (RQIKIYFQNRRMKWKK, SEQ ID NO: 58) composed of 16 peptides may be used (Joliot et al., Proc Natl Acad Sci USA 88: 1864-1868 (1991); Derossi et al., J Biol Chem 269, 10444-10450 (1994); Joliot, A. and A. Prochiantz, Nat. Cell Biol. 6(3): 189-96 (2004)), and in addition to the penetratin, peptide sequences having similar sequences and mechanisms are broadly called protein transduction domains (PTDs).
As another example, Antennapedia homeodomain-derived penetratin (RQIKIYFQNRRMKWKK) composed of 16 peptides may be used (Joliot et al., Proc Natl Acad Sci USA 88: 1864-1868 (1991); Derossi et al., J Biol Chem 269, 10444-10450 (1994); Joliot, A. and A. Prochiantz, Nat. Cell Biol. 6(3): 189-96 (2004)), and in addition to the penetratin, peptide sequences having similar sequences and mechanisms are broadly called protein transduction domains (PTDs). It was disclosed that a mechanism of intracellular material transduction of such a PTD was performed by disrupting a cell membrane on a cell surface and delivering a material into the cell, or by accumulating a material in an endosome through endocytosis caused by electrostatic interaction between a negative charge of various glycans of a membrane protein present on the cell surface and a positive charge of basic amino acid residues constituting the PTD to be delivered into the cell, not by directly permeating a material into the cell membrane (J. S. Wadia, et al., Nat. Med. 10: 310-315 (2004); I. Nakase, et. al. Biochemistry 46: 492-501 (2007); H. L. Amand, et al., Biochim. Biophys. Acta (2011)). However, such a material transduction mechanism of the PTD has difficulty in transducing materials such as peptides, proteins, nucleic acids, etc. into a deep tissue in a living body. Particularly, peptides or proteins are accumulated in an endosome, and when binding to lysosomes in the cell, easily degraded by a protease of the lysosome. Accordingly, when the peptides or proteins are transduced in a sufficiently high concentration using the PTD, an effective component capable of acting on a target in a cytoplasm can be emitted from the endosome, which becomes a problem occurring in the protein transduction using the PTD (F. Milletti, Drug Discovery Today 17: 850-860 (2012)).
Later, in the 2000s, MTS (AAVLLPVLLAAP; SEQ ID NO: 59) derived from a signal peptide of a fibroblast growth factor (FGF) was synthesized and manufactured (DaeWoong Jo et al., Nat. Biotech. Vol. 19, 2001). The MTS is composed of 12 hydrophobic amino acids, in which one valine and two leucines are present between a sequence continuously having two alanines, and a proline is included, and has quite different characteristics from the conventional PTD. In addition, recently, as a more improved transduction domain, a new cell membrane permeable peptide having more improved efficiency of delivering materials into cells than the PTD and MTS and different structural and electrostatic characteristics was developed, and the peptide was a macromolecule transduction domain (MTD) (refer to WO 2008/093982).
When the MTD, which was technology developed by the inventors, is used, unlike the transduction into an HIV-TAT cell, endocytosis and energy are not needed in the intracellular transduction of a material, and rigidity and integrity of the cell membrane for direct interaction with the cell membrane serve as important factors, and thus continuous transduction between cells can also be performed. For this reason, the MTD has a high efficiency of delivering a target protein into a cell, and enables deliver into a deep tissue in a living body, compared to TAT, which is a conventional cell membrane permeable peptide. In addition, a hydrophobic MTD derived from a signal sequence of a secretory protein or a cell membrane protein is manufactured by modifying a sequence of a hydrophobic region of a signal peptide largely composed of three parts including a hydrophobic region at an N-terminus and secreted-protein cleavage sites at a C-terminus, the hydrophobic region forming a helix structure to have a cell-membrane-targeting activity. The MTD is directly permeated through the cell membrane without damage to a cell, and allows macromolecules such as proteins to be delivered into the cell to exhibit their own functions.
However, since the MTD is usually composed of hydrophobic amino acids, and thus decreased in physical properties and availability, when manufactured in connection with a strongly-hydrophobic peptide or protein, the MTD may be precipitated when melted under a general buffer condition at a predetermined concentration or more, or according to a characteristic of a transducing material. Accordingly, combination with the MTD, which is not precipitated within a concentration range to be used and exhibits optimum permeability and activity in consideration of characteristics of a material to be transduced, may also be needed.
Therefore, the inventors developed a new cell membrane permeable domain having improved cell membrane permeability through deletion or modification on an amino acid sequence, or chimeric fusion, to improve physical properties and availability of the conventional MTD and increase cell membrane permeability, and confirmed an intracellular transduction effect thereof, thereby proving a more improved effect than the conventional MTD. Thus, the present invention was completed.